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Sample GSM1422963

Query DataSets for GSM1422963

Status

Public on Apr 24, 2017

Title

Hmel-shPTEN-H4K12AC

Sample type

SRA

Source name

Hmel-shPTEN

Organism

Homo sapiens

Characteristics

partially transformed melanocytic line: HMEL
clone subtype: Hmel-shPTEN
cell phentype: tumorigenic variant
chip antibody: H4K12AC
chip antibody vendor-cat.#: Active Motif-39165

Treatment protocol

Cells were infected with mko-shGFP or mko-shPTEN lentiviruses. Two hundred million cells were harvested for ChIP.

Growth protocol

Cells were maintained in standard tissue culture conditions (5% CO2 , 37oC) in DMEM medium supplemented with 10%FBS and Pen-Strep.

Extracted molecule

genomic DNA

Extraction protocol

Cells (5 million per antibody) were crosslinked using 1% paraformaldehyde for 10mins at 37 degrees. Reaction was quenched by 0.125M glycine for 5mins, cells washed with PBS and stored at -80oC. Next day cells were thawed on ice and lysed with RIPA buffer (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0, 140mM NaCl, 1% Triton x-100, 0.2%SDS, 0.1% DOC) for 10min on ice. Sonication was performed using Branson Sonifier 250 to achieve shear length of 200-500bp. Antibodies were bound to dynabeads for 1 hr at 4oC. Extracts were then incubated overnight with antibody-dynabead mixture Immunecomplexes were then washed 5 times with RIPA buffer, once with RIPA-500 (RIPA with 500mM NaCl) and once with LiCl wash buffer (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0, 250mM LiCl, 0.5% NP-40, 0.5% DOC). Elution and decrosslinking was performed in direct elution buffer (10mM Tris-Cl pH 8.0, 5mM EDTA, 300mM NaCl, 0.5% SDS) by incubating immunecomplxes at 65oC for 4-16hrs. Proteinase K (20mg/ml) and RNaseA treatment was performed and DNA cleaned up using SPRI beads (Beckman-Coulter).
Libraries were prepared using NEB reagents as described earlier (Garber et al 2012). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 12 cycles and library fragments of ~300-500 bp (insert plus adaptor and PCR primer sequences) were size selected by SPRI beads. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the HiSeq2000 following the manufacturer's protocols.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2000

Description

processed data file: Hmel-shPTEN_45_dense.bed

Data processing

ChIP-Seq reads were aligned using Bowtie (version 1.0.0) to human genome assembly NCBI Build 37 (UCSC hg19) with the following parameters: -n 1 -m 1 --best –strata (uniquely mapped reads with one mismatch were retained).
We used the ChromHMM method with default parameters to derive genome-wide chromatin state maps for all cell types. Sequencing reads that map to the same genomic location and strand were counted once in the input data in order to avoid biases due to PCR artifacts. This number of states was chosen, because it was the minimum number required to separate bivalent and poised states from active states. The 45 state model was learned jointly on all chromatin marks from H and Hmp. The chromatin state segmentation of Hmel-shGFP, Hmel-shPTEN, Pmel-shGFP and Pmel-shPTEN was produced subsequently by applying this model to the chromatin data from these cell types. Prior to that, we normalized each chromatin mark in Pmel-shGFP and Pmel-shPTEN to have the same number of binary calls across these two cell types in the binarization procedure of ChromHMM.
Genome_build: hg19
Supplementary_files_format_and_content: Gnome segmentation files generated for each cell type by ChromHMM.
Supplementary_files_format_and_content: *18_dense.bed files contain 18 state chromatin model data based on ChIP-Seq dataset for each individual cell line.

Submission date

Jun 30, 2014

Last update date

May 15, 2019

Contact name

Kadir Caner Akdemir

E-mail(s)

kcakedemir@mdanderson.org

Organization name

MD Anderson Cancer Center

Street address

1515 Holcombe Blvd